Value of Vitamin D Metabolite Ratios in 3 Patients as Diagnostic Criteria to Assess Vitamin D Status

Abstract Although clinical guidelines recommend measuring total plasma 25-hydroxyvitamin D (25[OH]D) to assess vitamin D (VitD) status, this index does not account for 3-fold inter-individual variation in VitD binding protein (VDBP) level. We present 3 individuals with total plasma 25(OH)D levels of 10.8 to 12.3 ng/mL (27-30.7 nmol/L). Because Endocrine Society guidelines define VitD deficiency as 25(OH)D ≤ 20 ng/mL (50 nmol/L), all 3 would be judged to be VitD deficient. VitD3 supplementation increased 25(OH)D to the range of 31.7 to 33.8 ng/mL (79.1-84.4 nmol/L). Patient #1 exhibited secondary hyperparathyroidism; VitD3 supplementation decreased parathyroid hormone (PTH) by 34% without a clinically significant change in PTH levels in the other 2 individuals. Thus, 25(OH)D level did not distinguish between the 1 patient who had secondary hyperparathyroidism and the 2 who did not. We therefore inquired whether VitD metabolite ratios (which are VDBP-independent) might distinguish among these 3 individuals. Of all the assessed ratios, the 1,25(OH)2D/24,25(OH)2D ratio was the most informative, which had a value of 102 pg/ng in the individual with secondary hyperparathyroidism but lower values (41 and 20 pg/ng) in the other 2 individuals. These cases illustrate the value of the 1,25(OH)2D/24,25(OH)2D ratio to provide clinically relevant information about VitD status.


Introduction
The Endocrine Society and The Institute of Medicine suggest measuring total plasma 25-hydroxyvitamin D (25[OH]D) to assess vitamin D (VitD) status but suggest different definitions of VitD deficiency and sufficiency [1,2].Recent publications question the value of VitD in preventing bone fracture in the general population [3].The existing controversy in the literature is due in part to the fact that total 25(OH)D measurement does not account for the 3-fold inter-individual variation in VitD binding protein (VDBP) levels [4].Although > 99% of circulating VitD metabolites are bound to proteins (albumin and VDBP), the biologically relevant form is the free fraction [5].Two enzymes play critical roles in VitD metabolism.1α-Hydroxylase converts 25(OH)D into 1,25-dihydroxyvitamin D (1,25[OH] 2 D)the biologically active form.24-Hydroxylase mediates metabolic clearance of 25(OH)D and 1,25(OH) 2 D by converting them to 24,25-dihydroxyvitamin D (24,25[OH] 2 D) and 1,24,25-trihydroxyvitamin D (1,24,25[OH] 3 D), respectively [6,7].Using ratios of VitD metabolites provides an indirect approach to account for VDBP levels [8][9][10].Tang et al suggested that the 1,25(OH) 2 D/24,25(OH) 2 D ratio is superior to total 25(OH)D to assess VitD status [10].This approach provides VDBP-independent indices that assess VitD status more accurately and can better predict clinical outcomes.Other VitD metabolite ratios have been suggested by others [8,11,12].We conducted a clinical trial in otherwise healthy individuals with 25(OH)D ≤ 20 ng/mL (≤ 50 nmol/L) studied before and after VitD3 supplementation [13].This study identified 3 individuals with total plasma 25(OH)D of 10.8 to 12.3 ng/mL (27-30.7 nmol/L), whom the Endocrine Society guidelines classify as VitD deficient and for whom they recommend VitD supplementation [1].When we assessed responses to VitD3 supplementation, one patient had what we propose to define as "overt VitD deficiency" in association with secondary hyperparathyroidism.One patient appeared to be VitD sufficient, and one appeared to have what we propose to label as "subclinical VitD deficiency" without secondary hyperparathyroidism.These cases illustrate the potential utility of VitD metabolite ratios in assessing clinical VitD status.

Case Presentations
All blood samples were obtained at ∼7 AM in the fasting state.The reference ranges for all lab values are listed in the tables.
Table 1 summarizes demographic and anthropometric characteristics.Table 2 summarizes lab results both before (baseline) and after VitD3 supplementation for all 3 patients.

Diagnostic Assessment
Although all 3 patients met Endocrine Society's criteria for VitD deficiency [1], only one had "overt VitD deficiency" in association with secondary hyperparathyroidism (as defined in the "Introduction" to this Case Report).One was VitD sufficient, and one had "subclinical" VitD insufficiency without secondary hyperparathyroidism (Table 3).Although VitD3 supplements decreased PTH levels by 34% in patient #1, patients #2 and #3 experienced only clinically insignificant 3% to 5% increases in PTH after VitD3 supplementationindicating the absence of secondary hyperparathyroidism at baseline.Total plasma 25(OH)D has limitations in assessing VitD status as it does not account for VDBP levels.Two individuals with the same total 25(OH)D level may have different free fractions depending on their VDBP levels.The use of VitD metabolite ratios provides a VDBP-independent index of VitD status [9,14].For example, Tang et al [10] 3).
We developed a mathematical model to estimate relative activity of the 24-hydroxylase enzyme, which catabolizes 25(OH)D and 1,25(OH) 2 D [15].Our model suggests that suppression of 24-hydroxylase activity provides the body's first line of defense to compensate for limited availability of VitD, resulting in slower metabolic clearance of 1,25(OH) 2 D and 25(OH)D.Secondary hyperparathyroidism, which develops in more severe VitD deficiency, leads to increased production of 1,25(OH) 2 D-the biologically active metabolite.The 1,25(OH) 2 D/24,25(OH) 2 D ratio incorporates the 24,25(OH) 2 D level in the denominator -thereby providing information about 24-hydroxylase activity.Our model estimated a ∼10-fold dynamic range for 24-hydroxylase activity as a function of 25(OH)D levels.Maximum and minimum suppression of 24-hydroxylase occurs when 25(OH)D is < 10 to 20 ng/mL (< 25-50 nmol/L) or > 50 ng/mL (125 nmol/L), respectively.Our mathematical model estimated that Case #1 (who had secondary hyperparathyroidism) exhibited only 15% of maximal 24-hydroxylase activity.Case #2, whom we classified as VitD insufficient, and case #3, whom we classified as VitD sufficient, exhibited

Outcome and Follow-Up
VitD3 supplementation increased total levels of 25(OH)D from the range of 10.

Discussion
Physiologists recognize that homeostatic regulatory mechanisms maintain healthy physiology [16,17].Endocrinologists leverage this notion to diagnose overproduction and underproduction of hormones (eg, measuring thyrotropin [TSH] to diagnose thyroid function) [18].Endocrinologists apply the "free hormone hypothesis" to account for the impact of binding proteins on  measured hormone levels.For example, pregnancy and estrogens increase thyroxine binding globulin levels thereby increasing total thyroxine levels [19].Unfortunately, these concepts have not yet been incorporated into routine clinical assessment of VitD status.Because of inter-individual variation in VDBP levels, it is critical to account for this potential confounder [9,14].When treating VitD deficiency in association with secondary hyperparathyroidism, documentation of a decrease in PTH confirms that homeostatic mechanisms had been triggered prior to VitD3 supplementation.However, our mathematical model and the existing literature suggest that secondary hyperparathyroidism does not develop until VitD deficiency becomes relatively severe [2,20,21].Nevertheless, even in milder VitD deficiency and in the absence of secondary hyperparathyroidism, the body triggers another homeostatic mechanism to maintain 1,25(OH) 2 D levels in the physiological range.Suppression of the 24-hydroxylase enzyme provides the first line of defense and is deployed at milder stages of VitD deficiency.This provides a rationale to incorporate 24,25(OH) 2 D measurement in assessing VitD status-especially, to differentiate patients with secondary hyperparathyroidism from patients with subclinical VitD deficiency but normal PTH levels.

Learning Points
• Total plasma 25(OH)D does not account for the confounding effect of inter-individual variation in VDBP levels.• VitD metabolite ratios provide VDBP-independent indices of VitD status.• The body compensates for VitD deficiency by (a) suppression of 24-hydroxylation and (b) triggering secondary hyperparathyroidism. • Ratios that incorporate 24,25(OH) 2 D levels reflect 24-hydroxylase activity.• Preliminary evidence suggests that the 1,25(OH) 2 D/ 24,25(OH) 2 D ratio may provide an index that identifies patients most likely to exhibit secondary hyperparathyroidism.
The 1,25(OH) 2 D/ 24,25(OH) 2 D ratio differentiated between patients #2 and #3 who had similar baseline total 25(OH)D levels.The 1,25(OH) 2 D/24,25(OH) 2 D ratio classified patient #2 as VitD insufficient and patient #3 as VitD sufficient.The use of VitD metabolite ratios, particularly the 1,25(OH) 2 D/24,25(OH) 2 D ratio, has 2 advantages over the traditional approach (total 25[OH]D measurement): accounting for VDBP levels and providing information about the body's response to VitD status as reflected by 24-hydroxylase activity.Notwithstanding the potential value of VitD metabolite ratios, we recognize the challenges in implementing a major change in diagnostic criteriaincluding, the need to assure standardization, harmonization, and quality control in the assays for 25(OH)D, 1,25(OH) 2 D, and especially the less widely available assay for 24,25(OH) 2 D.